Identifying Dnarrr proteins by complementation of E coli mutants

Most of our knowledge of eubacterial DNA-RRR pathways is based on E. coli (CameriniOtero and Hsieh 1995; Kowalczykowski 2000). Plastids are probably descendants of an ancient cyanobacterium (Martin et al. 2002; Chapter 1) and have retained components of eubacterial DNA-RRR pathways including homologues of DNA polymerase, gyrase, and RecA (Table 2). In some cases, these plastid proteins have been shown to complement mutations in the homologous

Exonuclease. Helicase

RecA

DNA Polymerase

Branch Migration Proteins

Double Holliday Junction Resolvase

Non Recombinant Cross-over

Patched Spliced

Fig. 15. Double-strand-break model of homologous recombination showing main proteins involved (Szostak et al. 1983; Kowalczykowski 2000).

E. coli proteins (Cho et al. 2004). Complementation of E. coli mutations provides a functional assay for identifying cDNAs encoding plastid-DNA RRR proteins andwould appear to be an attractive method for isolating plant homologues of E. coli DNA-RRR proteins. Using cDNA libraries, plant cDNAs that complement a number of E. coli mutations in DNA-RRR genes were isolated (Pang et al. 1993b) including RecA (Pang et al. 1992), and ruvC and recG mutants (Pang et al. 1993a). Unfortunately, the isolated cDNAs did not encode proteins related to characterized DNA-RRR proteins hindering progress. One of the isolated cDNAs complementing ruvC and recG double mutants was subsequently identified as plastocyanin (acc number P42699) raising questions on the validity of these library screening experiments. Screening cDNA libraries for functional complementation of E. coli mutants is technically difficult especially if complementation is not strong. Pitfalls include transformation or transfection of the tiny number of E. coli cells in which the mutation has reverted or been suppressed, which will give rise to viable cells on antibiotic medium. Alternatively, the cDNA may encode a protein that rescues the mutation indirectly, for example, by stabilising a temperature sensitive E. coli mutant protein.

Exonuclease. Helicase

DNA Polymerase

Branch Migration Proteins

Double Holliday Junction Resolvase

Non Recombinant Cross-over

Patched Spliced

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